Non-Instrumented Incubation of a Recombinase Polymerase Amplification Assay for the Rapid and Sensitive Detection of Proviral HIV-1 DNA Lorraine Lillis1, Dara Lehman2, Mitra C. Singhal1, Jason Cantera1, Jered Singleton1, Paul Labarre1, Anthony Toyama1, Olaf Piepenburg3, Mathew Parker3, Robert Wood4, Julie Overbaugh2, David S. Boyle1* 1 PATH, Seattle, Washington, United States of America, 2 Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 3 TwistDx Limited, Minerva Building, Babraham Research Campus, Babraham, Cambridge, United Kingdom, 4 Department of Atmospheric Sciences, University of Washington, Seattle, Washington, United States of America
Abstract Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25–43uC, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10uC and rarely above 43uC. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31uC and 43uC. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30uC. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10uC and 30uC. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15uC, 20uC, 25uC and 30uC. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in LRS. Citation: Lillis L, Lehman D, Singhal MC, Cantera J, Singleton J, et al. (2014) Non-Instrumented Incubation of a Recombinase Polymerase Amplification Assay for the Rapid and Sensitive Detection of Proviral HIV-1 DNA. PLoS ONE 9(9): e108189. doi:10.1371/journal.pone.0108189 Editor: Cecilio Lo´pez-Galı´ndez, Centro Nacional de Microbiologı´a - Instituto de Salud Carlos III, Spain Received April 29, 2014; Accepted August 20, 2014; Published September 29, 2014 Copyright: ß 2014 Lillis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: This work was supported by the United States National Institutes of Health, National Institute of Allergy and Infectious Diseases (http://www.niaid.nih. gov/Pages/default.aspx)via award number R01AI097038. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: OP and MP are employees are of TwistDx Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors have no competing interests. * Email: [email protected]
as p24 antigen detection or reverse transcriptase activity [2]. However, the infrastructure and equipment requirements to perform PCR-based HIV diagnostics results in the need to ship specimens from rural clinics to centralized facilities for testing. In low income countries, shipping and the logistics of reporting results can delay test results for months, often contributing to loss to follow up of confirmed HIV infection [3]. The outcome for HIV infected infants who are left untreated is very poor and over 50% of HIV infected infants die within 2 years if left untreated [4]. By contrast, rapid treatment can potentially lead to a reduced viral reservoir and better outcomes, further emphasizing the need for rapid point of care diagnostics [5]. In the last decade, an expanding variety of novel NAATs have been developed to specifically meet the challenges of performing diagnostics outside of well-equipped facilities in low resource settings (LRS) [1,6–9]. A particular area of research has been the
Introduction The application of nucleic acid amplification technologies (NAAT) has had a significant and beneficial impact on the diagnosis of infectious diseases. While a wide variety of molecular based technologies have been developed to accurately detect a broad range of clinically relevant pathogens in just a few hours [1], the majority of these are designed for use in fully equipped laboratories with good infrastructure, reliable electrical supply and with highly trained staff to operate them [2–7]. Therefore, there remains a critical need for high quality diagnostics that can be performed closer to the point of care in low resource settings (LRS) to aid in the clinical diagnosis and subsequent treatment of endemic diseases such as HIV, tuberculosis and malaria. PCRbased assays are typically used for early infant diagnosis (EID) of HIV, as they are more sensitive than other existing methods such PLOS ONE | www.plosone.org
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recently a group reported the sensitive detection of Plasmodium falciparum using an RPA assay and minimal instrumentation [29]. The use of ambient temperature or simple chemical heaters for an RPA-based diagnostic has not yet been evaluated. We previously described the development of an RPA assay for rapid diagnosis of infant HIV that showed high sensitivity and specificity to the major subtypes of HIV-1 circulating globally using electrical powered, laboratory-based instrumentation [17]. In this study we investigated the potential for ambient temperatures to incubate this HIV-1 RPA assay in the absence of a reliable power source. We also analyzed meteorological data to understand the seasonal temperature fluctuations across sub Saharan Africa, the primary target market of our intended HIV diagnostic, to better understand if ambient temperatures could reliably support RPA assay use across this region. In addition, to incubate RPA assays when ambient temperatures are below 30uC, we developed a simple, low cost ($0.10 US) and reusable prototype chemical heater. Our findings in this study suggest that it is possible to develop affordable and effective incubation platforms for an RPAbased diagnostic for use in the most resource limited of settings.
development of isothermal amplification methods for nucleic acid based diagnosis. These technologies employ a uniform temperature to amplify targeted nucleic acids [1]. When compared to current PCR assays, the use of isothermal technologies reduces the need for high precision instrumentation, consistent electrical power and complex sample preparation [10,11]. Such amplification methods include loop mediated amplification (LAMP) [12], nucleic acid sequence based amplification (NASBA) [13], cross priming amplification (CPA) [14], helicase dependent amplification (HDA) [15] and recombinase polymerase amplification (RPA) [16]. In support of these amplification technologies, several electricity dependent instruments are now available to incubate the assays and detect amplification [6,18–22]. Although some of these are battery powered, they still require some degree of sustainable electrical power for recharging, and their cost (while much less than large or mid-sized real time PCR instruments) may limit their widespread adoption for use in many peripheral settings. Cheap but robust incubation systems, such as the noninstrumented incubation amplification (NINA) platform, employ chemically derived heat for assay incubation; greatly reducing costs and increasing the potential for use at the periphery [11]. These platforms have been demonstrated to effectively amplify LAMP and HDA assays [23–25]. Detection of amplification products with NINA relies upon visual inspection of the test reaction for reactivity or the use of a sealed immunochromatographic strips to capture and detect hapten labeled amplicons [26– 28]. Simple yet effective technologies like these enhance the potential to use isothermal assays and introduce high performance NAATs to the most austere environmental settings where electrical power is often sporadic or absent. RPA is an isothermal amplification method that utilizes a recombinase to facilitate the insertion of oligonucleotide primers into their complement in a double-stranded DNA molecule [16]. The use of opposing primers allows the exponential amplification of a defined region of DNA in a manner similar to PCR. Detection of HIV RPA amplicons relies on the use of an oligonucleotide probe with a specific abasic nucleotide analogue that is recognized and cleaved by an endonuclease only when the probe is bound to its complementary target sequence [16]. The amplified products can typically be detected after only 5–20 minutes, depending on amplicon size and the template copy number and also on the sensitivity of the detection method [16]. This time to test result is significantly more rapid than other isothermal assays which generally have incubation times in the range of 45–60 minutes [12–15]. RPA is also unique compared to many other isothermal amplification methods in that it has an incubation temperature range similar to ambient temperatures ranges found south of 20uN in continental Africa and has been reported to amplify DNA across a broad range of incubation temperatures ranging from 25– 43uC using the standard reaction conditions and formulation [16]. Moreover, in contrast to the other isothermal NAAT technologies, RPA does not primarily rely on physio-chemical dependent hybridization of oligonucleotides for functionality, but rather operates by an enzymatically driven primer-binding process. For this reason RPA does not require the reaction temperature to be precisely controlled during the reaction to be specific and an assay will tolerate comparatively large variance in the incubation temperature without apparent loss of overall sensitivity or specificity. These features translate to reduced power requirements and less stringent temperature control, further reducing the complexity and cost of an incubator. The use of simpler heating devices has been explored in previous studies. For example, PLOS ONE | www.plosone.org
Materials and Methods RPA amplification and Detection TwistAmp nfo RPA reactions were supplied by TwistDx Ltd., Cambridge, United Kingdom. HIV pol RPA primers and probe, described previously [17], were added to the RPA nfo assays. 10 copies of HIV DNA were added to each of 3 replicate reactions at each temperature tested. HIV proviral target DNA was from the ACH-2 cell line that contains a single full-length integrated copy of HIV-1 Bru (clade M, subtype B; GenBank accession number K02013.1) DNA per cell [30] and was prepared and quantified as previously described [17]. Negative template controls (NTC) contained nuclease free water in place of DNA. All TwistAmp reaction mixtures were prepared in a final volume of 50 mL as previously described [17]. Incubation was performed at the test temperature for either 20 or 30 minutes, with brief mixing by inverting 3 times at 5 minutes. The RPA nfo reactions were immediately placed on ice to slow the reaction and limit the effect of the slight differences in incubation times before the sequential addition of 5 mL EDTA (250 mM) to halt each reaction. Detection of hapten-labeled RPA amplicons was assessed with an immunochromatographic strip (ICS) fully enclosed in a cassette (BESt Cassette II; BioHelix Corp., Beverly, MA) [31]. Amplification products were also analyzed by gel electrophoresis.
RPA incubation To mimic various ambient temperatures, all test reaction components, RPA reagents, aluminum blocks machined to hold 0.2 mL PCR tubes, and sodium acetate trihydrate (SAT) heaters were pre-incubated for 2 hours prior to reaction set up in an environmental control chamber (Coy Laboratory Products Inc., MI, USA; or Associated Environmental Systems, Ayer, MA, USA) at temperatures between 10–44uC. Three thermocouples in each environmental chamber confirmed the temperature prior to reaction set up. Humidity in all experiments was at levels between 60–65%. The controlled incubation of RPA assays was performed using a conventional PCR thermocycler (Applied Biosystems, Foster City, CA, USA).
Exothermal heaters Sodium acetate trihydrate (SAT) (Sigma Aldrich, St Louis, MO, USA) mixtures at concentrations of 70%, 72.5% and 75% were prepared and liquefied by heating to 70uC, and aliquoted into 2
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either 15 mL screw cap tubes (Fischer Scientific, Pittsburg, PA, USA) or 22 mL lidded plastic souffle´ cups (Dart Container Corporation, MI, USA) with insulating foam (McMaster-Carr, Elmhurst, IL, USA) cut to fit each receptacle (Figure 1). A 5 mm diameter punch was made in the center of each cup lid to allow the insertion of a 0.2 mL PCR tube containing RPA reactions. Heat release via crystallization of the liquid SAT mixtures was induced by seeding with powdered SAT. Solidified SAT reactors were heated to 80uC for 20 minutes to reliquefy the SAT mixture, by either placing into a water bath or incubator heated to 80uC.
Results HIV-1 RPA assay performance at temperatures from 15uC to 44uC To determine the range of temperatures that our HIV-1 RPA assay can tolerate, we tested the ability of this specific RPA assay to amplify 10 copies of HIV DNA at a range of temperatures from 15uC to 44uC using ICS cassettes to detect RPA amplicons. Initially, we assessed this temperature range under precisely controlled conditions using a thermocycling machine. For assays incubated for 20 minutes at #30uC, performance of the HIV-1 RPA assay on 10 copies of HIV-1 proviral DNA was not optimal, with only 3 temperatures producing any positive results (1/3 at 15uC and 29uC, or 2/3 at 30uC), and all 3 replicates failing at temperatures between 20uC and 27uC (Table 1). The lower incubation temperature reduces enzyme activity leading to limited amplicon formation after 20 minutes and is not be enough to be detected by LFS; however as shown by the 1 positive sample in the reactions incubated at 15uC there is remains an occasional possibility of sufficient product being amplified for detection. When incubation times were increased to 30 minutes, performance improved at temperatures between 27uC and 30uC, with all 3 replicates being positive for each temperature, but not at temperatures of 25uC and below (data not shown). Even longer incubation times than 30 minutes, which may have resulted in positive RPA signals at low temperatures, have not been tested in this study. Incubation temperatures between 31uC to 43uC provided optimal conditions in which all 3 replicates were positive at each of the 8 temperatures tested (24/24, 100%; Table 1). When increased to 44uC, performance was impaired with only 1 of 3 reactions positive. There were no false positives at any of the temperatures tested (0/15, Table 1). Gel electrophoresis was also performed on reactions from each temperature (Data not shown). Amplification products of the expected size (,150 bp) were observed in all positive reactions heated at temperatures from 27uC–44uC. No amplification products were observed in reactions that gave negative results incubated below 27uC. This indicates that the negative results observed at lower temperatures were due to limited or no production of RPA amplicons presumably due to reduced enzyme activity and not due to competition from nonspecific product formation. We next tested our HIV RPA assay using an environmental chamber to simulate reactions incubated at ambient temperatures between 15uC and 44uC. Results were very similar to the thermocycler incubation results. Incubation for 20 minutes at temperatures #30uC were suboptimal, with only 2 of 3 reactions at 29uC, and 0 of 3 reactions positive at all other temperatures # 30uC (Table 1). When incubation times were increased to 30 minutes, 3 of 3 reactions performed at 30uC were positive, with all other results unchanged (data not shown). Incubation at ambient
Temperature measurement Ambient temperatures and thermal profiles within the SAT solutions were measured using T-type – Physitemp IT-23 thermocouples (Omega Engineering, Everett, WA, USA) and a NI 9211 data logger (National Instruments, Redmond, WA, USA). Prior to use, each thermocouple was calibrated in boiling distilled water to verify a 100uC temperature reading on the data logger. For ambient temperature assessment within the environmental chambers, one thermocouple was positioned beside the 0.2 mL reaction tube within the tube rack, and one next to the tube rack. As a proxy for reaction temperature, an additional thermocouple was inserted through a 1.27 mm diameter hole in the lid of a 0.2 mL PCR tube filled with 50 mL deionized water which represents the RPA reaction volume. When incubating RPA reactions in the SAT heaters, a thermocouple was positioned beside the 0.2 mL reaction tube (Figure 1). The temperature was recorded at a rate of 1 data point per second for the duration of each experiment. Temperature data was further analyzed and displayed using Microsoft Excel (Microsoft Corp., Redmond, WA, USA).
Air temperature data A previously published bias-corrected meteorological dataset for Africa was analyzed to determine the range of daily ambient temperatures expected [32]. The dataset is derived from meteorological analyses of the physical state of the atmosphere that are produced every three hours using a weather forecasting model. The horizontal resolution of the dataset is 161 degree latitude/longitude (approximately 1006100 km) [33]. Air temperature data are bias and elevation corrected with qualitycontrolled meteorological data from stations in Africa, and the diurnal cycle of surface air temperature is adjusted to provide the best match with meteorological station data [32]. These corrections remove a bias of 2–4uC in surface air temperature over the African continent. The dataset is available for the years 1948– 2010, but we use data only from the period 2000–2010 because the statistics were found to be insensitive to the inclusion of additional years.
Figure 1. The prototype SAT heater used to incubate RPA reactions. (A) A photograph of an activated SAT heater, the reaction tube is filled with blue dye to improve definition. (B). A cross sectional diagram depicting the SAT heater and other test components. doi:10.1371/journal.pone.0108189.g001
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PLOS ONE | www.plosone.org 2/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3
30
31
33
35
37
39
40
42
4 0/1
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
0/3
0/3
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
209–20 minutes total incubation time; 309–30 minutes total incubation time; NTC - no template control. doi:10.1371/journal.pone.0108189.t001
1/3
1/3
29
44
0/3
27
3/3
0/3
25
43
0/3
20
0/1
309
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
2/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
0/3
0/3
0/3
0/3
0/3
0/3
209
2/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
2/3
0/3
0/3
0/3
0/3
309
209
309
209 0/3
HIV
NTC
HIV
1/3
Ambient Temperature
Thermocycler
15
Temp (6C)
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
209
NTC
Table 1. A comparison of the performance of HIV RPA assays with reaction incubation in either a thermocycler or via ambient air temperature.
309
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
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and 75% SAT heaters, with and without insulation, to incubate HIV RPA assays from ambient temperatures of 10uC, 15uC, 20uC, 25uC and 30uC. Nine of 10 of the insulated reactions reached 95% of the maximum recorded incubation temperature in under 15 seconds (range 4 to 18 seconds, Table 2). The noninsulated reactions took longer to reach the maximum temperatures recorded, with a range of 5 to 85 seconds. At ambient temperatures of 15uC and above, insulated 75% SAT heaters rapidly generated sufficient heat to provide incubation for 30 minutes maintaining the 31–43uC temperature range required for the HIV-1 RPA assay (Table 2). At the lower ambient temperature of 10uC, the temperature fell to below 30uC in less than 10 minutes (Table 2). The 72.5% SAT heater was comparable above at ambient temperatures above 15uC, but again also did not maintain the minimal required temperature of 31uC after 20 minutes at ambient temperatures #15uC. Insulated SAT reactors containing 72.5% and 75% SAT were then assessed for their ability to incubate the HIV RPA assays at a range of ambient temperatures from 10uC–30uC in 5uC increments. As before, triplicate HIV-1 RPA assays were performed on 10 copies of HIV-1 DNA at each starting ambient temperature and SAT concentration. All 12 of 12 (100%) reactions at ambient temperatures between 15uC and 30uC were positive when incubated by either activated SAT mixture for 20 minutes, while no false positives were observed (Table 3). Starting at an ambient temperature of 10uC, all triplicate reactions failed when incubated with either the 72.5% or 75% SAT heater (Table 3). However, when repeated with increased incubation times of 30 minutes, to compensate for reduced enzyme activity at suboptimal temperatures, all 3 reactions starting at ambient temperature of 10uC were positive with either concentration of SAT tested (Table 3).
temperatures between 31uC and 43uC produced consistent results with all replicates positive in this temperature range (24/24, 100%). At 44uC, performance was once again suboptimal, with only 2 of 3 reactions positive (Table 1).
A non-instrumented heat source for incubation of RPA when ambient temperatures are #30uC The previous data suggests that an external heat source is necessary for the HIV-1 RPA assay used in this study when the ambient temperature is below 30uC. We investigated the potential to use a rechargeable, exothermic material commonly used in hand warmers, a mixture of sodium acetate trihydrate (SAT) and water. A variety of SAT concentrations (from 50% to 80% w/v in water) were assessed for the temperature profiles produced after their activation from an ambient temperature of 22uC (Table S1). This preliminary screen established that SAT concentrations of 70%–75% produced heat profiles that were above 31uC but did not exceed 45uC. Next, simulated assay temperature was measured for 30 minutes after activation of 8 mL volumes of 70%, 72.5% and 75% SAT solutions that had been stored for $2 hours prior to activation at ambient temperatures of 10uC, 15uC, 20uC, 25uC and 30uC (Figure 1). Each SAT solution was activated 32 times in individual tests at all of the ambient temperatures under consideration with average peak temperatures produced as shown in Figure 2. Maximal temperature was generally reached within 1 minute of the crystallization of the SAT mixture. The range of temperatures generated by each mixture did not exceed 45uC even when the heaters were conditioned to a simulated ambient temperature of 30uC prior to activation. However, when the ambient was reduced to 10uC, the 70% mixture failed to reach 30uC and was therefore not tested for incubation of RPA reactions (Figure 2). To test the ability to use SAT heaters to incubate the HIV-1 RPA reaction, we identified small plastic tubs that allow the RPA reaction mixtures to be placed close to the center of the activated SAT mixture (Figure 1). We assessed the performance of 72.5%
Analysis of daily and seasonal temperature data from Sub Saharan Africa To assess the viability of using RPA assays under ambient meteorological conditions, we analyzed an existing dataset of the
Figure 2. Median peak temperatures produced by different concentrations of SAT when held and activated under a variety of ambient temperatures. Ambient temperatures of SAT mixtures during activation: blue 10uC; green 15uC; orange 20uC; turquoise 25uC and red 30uC; SAT, Sodium acetate trihydrate. doi:10.1371/journal.pone.0108189.g002
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PLOS ONE | www.plosone.org 31 29 27 6 10 4
Maximum Temp. (uC)
Temp. (uC) at 10 minutes
Temp. (uC) at 20 minutes
Temp. (uC) at 30 minutes
Temp. loss over 30 minutes (uC)
Time (sec) to reach max. temp.
Time (sec) to 95% of max. temp.
No
8
82
22
13
17
26
35
18
123
9
28
31
34
37
Yes
15 No
11
89
18
21
22
30
39
5
94
5
32
34
36
37
Yes
20 No
15
57
16
24
27
33
40
5
123
3
37
38
39
40
Yes
25 No
5
111
10
31
34
38
41
4
14
3
38
39
40
41
Yes
30 No
85
287
6
37
40
42
43
6
80
8
25
27
31
33
Yes
10
7
52
21
13
17
25
34
No
75% SAT
12
109
7
30
32
35
37
Yes
15 No
7
53
16
18
20
26
34
5
111
5
35
37
39
40
Yes
20
6 3/3 3/3 3/3 3/3
15
20
25
30
309
3/3
3/3
3/3
3/3
3/3
209
0/1
0/1
0/1
0/1
0/1
309
0/1
0/1
0/1
0/1
0/1
209
3/3
3/3
3/3
3/3
0/3
SAT, Sodium acetate trihydrate; 209–20 minutes total incubation time; 309–30 minutes total incubation time; NTC - no template control; NTC, no template control. doi:10.1371/journal.pone.0108189.t003
209 0/3
Temperature (uC)
10
HIV
HIV
NTC
75% SAT
72.5% SAT
3/3
3/3
3/3
3/3
3/3
309
Table 3. The performance of HIV RPA assays when incubated in SAT reactors at a range of ambient temperatures from 10uC to 30uC.
SAT, Sodium acetate trihydrate. doi:10.1371/journal.pone.0108189.t002
Yes 33
Insulation
10
Ambient storage temp. (6C)
72.5% SAT
No
10
71
14
24
27
32
38
0/1
0/1
0/1
0/1
0/1
209
NTC
4
20
4
39
40
41
43
Yes
25
Table 2. Thermal profiles of the heat generated via the insulated and non-insulated SAT heaters under a range of ambient storage temperatures.
No
39
240
7
30
32
35
37
0/1
0/1
0/1
0/1
0/1
309
5
182
2
41
42
43
43
Yes
30 No
28
238
5
36
38
40
41
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surface air temperature in Africa between 2000–2010 [32]. Here we focus on temperatures recorded in the region of sub Saharan Africa (continental Africa south of 20uN) most afflicted by the HIV epidemic. Air temperature at any given site experiences significant variability on diurnal (day-night) to seasonal timescales. We distilled the data into 6 hourly intervals (0–6, 6–12, 12–18, 18– 24 hour local time) and examine statistics for each of four seasons (Dec-Feb, Mar-May, June-Aug, Sept-Nov). For each 161u location in the dataset, the season with the warmest mean temperature is termed the warm season and the season with the coldest mean temperature is termed the cold season. The RPA assay tests were shown to work well for the temperature range 30uC–43uC. Air temperatures in sub Saharan Africa were found to rarely exceed the upper limit (43uC) even during the warm season and the afternoon (12–18 hr local time; Figure 3A). For example, in the harshest desert regions north of 10uN, mean afternoon temperatures are below 43uC on over 70% of days. South of 10uN, where the HIV incidence is greatest and population density is higher, afternoon warm season air temperatures are lower than 43uC on over 95% of days. We examined the frequency at which the mean afternoon temperature exceeds 30uC during both the cold and warm seasons. During the cold season, the frequency of mean afternoon temperatures exceeding 30uC is lower than 10% in most locations. Exceptions include the desert regions north of 10uN where mean afternoon temperatures exceed 30uC on more than 50% of days during the cold season, and in the low-lying rainforest of the Congo Basin where mean afternoon temperatures exceed 30uC on 20–40% of days. During the warm season, a similar geographical pattern exists, but the frequency with which mean afternoon temperatures exceed 30uC is increased everywhere (Figure 3B). North of the equator during the warm season, mean afternoon temperatures exceed 30uC on over 50% of days. For most noncoastal locations south of 10uS, warm season mean afternoon temperatures exceed 30uC on approximately 60% of days. In the Congo Basin, warm season mean afternoon temperatures exceed 30uC on more than 40% of days. Exceptions include strongly elevated terrain (.2000 m) where mean afternoon temperatures rarely exceed 30uC. Analysis of the surface temperature data show that the mean afternoon air temperatures over most of subSaharan Africa exceed 10uC on nearly all days, even during the cold season (Figure 3C), so that a simple heating system should lead to successful RPA tests at most locations under almost all ambient environmental conditions.
Discussion RPA provides an attractive method for use in point of care diagnostics because it can be performed over a relatively broad range of incubation temperatures with no observed loss in performance when end point detection of the assay product was used. In this work we assessed the performance of a previously described HIV-1 RPA assay to reliably detect 10 copies of HIV DNA using ambient temperatures between 30uC–43uC, using the standard, commercially available formulation of RPA reagents. At ambient temperatures below 31uC, an exothermic chemical heater could be used to provide supplementary heat for optimal RPA performance to as low an ambient temperature of 10uC. These results demonstrate that, even in the absence of electricity or a battery dependent heater, RPA is significantly faster than other isothermal assays that are being developed as tools for NAAT based diagnostics in LRS. The incubation time required was only 20 minutes at the higher temperatures, with an increase to 30 minute incubations necessary at the lower temperature of 30uC to compensate for reduced enzyme kinetics at suboptimal temperatures. Longer incubation times have been shown to produce positive results at incubation times below 30uC when used with other unrelated RPA assays but longer incubation times were not tested in this study as this would lead to a less acceptable time to result. To reinforce the plausibility of using ambient temperature for RPA incubation we performed an analysis of existing meteorological data for the sub Saharan Africa region, the center of the HIV epidemic, to determine the daily ambient temperature ranges in the warmest and coldest seasons. In the warm season, the entire region has surface temperatures that often exceed 30uC after noon, providing potential to incubate RPA assays without the need for a heater during this season in many areas within this region. In addition, pooled temperature data revealed that average temperatures were rarely observed to elevate above 43uC in this region, and therefore temperatures too high for optimal RPA are not commonly encountered. However, the analysis of seasonal meteorological data highlighted that many regions in sub Saharan Africa have ambient temperatures below 30uC, especially in elevated regions which represent a significant area of this landmass. From analysis of the meteorological data we also noted that ambient temperatures of less than 10uC are not commonly observed in this region. While this work focused on sub-Saharan Africa where the HIV epidemic is greatest and so is the primary intended market for the HIV diagnostic, there are also other LRS worldwide which would
Figure 3. Maps depicting sub Saharan Africa and the frequency (fraction of days) on which mean afternoon (12–18 hr local time) surface air temperatures are below 436C during the warm season (A), exceed 306C in the warm season (B) and are above 106C in the cold season (C). doi:10.1371/journal.pone.0108189.g003
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greatly benefit from better access to RPA based NAATs. While the climate might differ to those found in Sub Saharan Africa, many will have day time ambient temperatures that will allow for incubation using the approaches discussed herein. To accommodate the lower temperatures observed we developed basic chemical heaters that can successfully incubate HIV-1 RPA assays even when the ambient temperature is as low as 10uC. These prototype SAT heaters were rechargeable by heating at 80uC for 20 minutes and cost less than $0.10 (USD) to make per prototype device. In areas where the temperatures fall below 10uC the SAT concentrations may be further adjusted to create a heater that will incubate the RPA reactions to within the required temperature ranges. Insulation of these heaters will be vital to minimize significant heat loss. For those with ambient temperatures above 45uC we would recommend that testing is held off until temperatures lie within in a more acceptable range. The SAT heater used in this study is only a prototype, and not the final design. It is anticipated that the final SAT heater device will be fully enclosed, with metal kinetic switch housed within the solution to initiate activation. The device can then be easily placed in a cup of boiled water to resolubilize the SAT before it is ready to be used again. Ambient temperatures and SAT heaters are not the only instrument free options available. In addition, it is interesting to note that human body temperature is within the optimal temperature range for our HIV-1 RPA assay, and suggests that manual incubation may be possible. A simple device could be created, which could be placed under the arms or held to incubate the tube to approximately 35–37uC and allow for amplification to occur. Overall, there is compelling evidence that RPA can be used for the application of high performance NAAT diagnosis of HIV-1 as well as other infectious diseases using either the ambient environmental temperature if above 30uC, or by using the most basic of heat sources for incubation when the ambient temperature is greater than 10uC. NAAT-based diagnostic tests in low resource settings face a variety of challenges, including access to adequate electrical power to run equipment or to recharge batteries, as many of these NAATs require prolonged incubation at elevated
temperatures (.55uC) within a narrow range for optimum amplification. Typically this means that powered instrumentation is necessary to address precise incubation temperatures. However RPA does not have such stringent requirements. A previously described RPA assay to detect methicillin resistant Staphylococcus aureus was similarly noted to detect target DNA in a temperature range spanning from 25uC to 44uC, [16], therefore it is highly probable that this temperature range could be applied to any RPA based target. This relatively broad range of incubation temperatures suggests that RPA can be effectively performed using either ambient temperatures without a heat source or by using a simple electricity-free heat source supplied via an exothermic chemical reaction [29]. Here we demonstrate the potential benefit of using RPA in point-of-care diagnostics in settings where access to reliable electrical power is limited. As this technology does not require a precise temperature for incubation, ambient temperatures or a simple heating device can be used to achieve accurate and reproducible results, making it a powerful diagnostic tool.
Supporting Information Table S1 Thermal profiles from preliminary screening of SAT concentrations of 50%–80% (w/v) after activation from an ambient temperature of 226C. Reactions were performed in insulated 50 mL tubes. (DOCX)
Acknowledgments We thank Dr Angelika Niemz for critical edits and comments during the drafting of this manuscript.
Author Contributions Conceived and designed the experiments: DB DL OP MP LL MS JC JS RW AT. Performed the experiments: LL MS JC JS RW AT. Analyzed the data: DB DL OP MP LL MS JC PL JS RW AT JO. Contributed reagents/ materials/analysis tools: OP MP DL JO RW. Wrote the paper: LL DB DL JC MC RW JO PL JS.
References 11. LaBarre P, Hawkins KR, Gerlach J, Wilmoth J, Beddoe A, et al. (2011) A simple, inexpensive device for nucleic acid amplification without electricitytoward instrument-free molecular diagnostics in low-resource settings. PLoS One 6: e19738. 12. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, et al.(2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28: E63. 13. Compton J (1991) Nucleic acid sequence-based amplification. Nature 350: 91– 92. 14. Fang R, Li X, Hu L, You Q, Li J, et al. (2009) Cross-priming amplification for rapid detection of Mycobacterium tuberculosis in sputum specimens. J Clin Microbiol 47: 845–847. 15. Vincent M, Xu Y, Kong H (2004) Helicase-dependent isothermal DNA amplification. EMBO Rep 5: 795–800. 16. Piepenburg O, Williams CH, Stemple DL, Armes NA (2006) DNA detection using recombination proteins. PLoS Biol 4: e204. 17. Boyle DS, Lehman DA, Lillis L, Peterson D, Singhal M, et al. (2013) Rapid detection of HIV-1 proviral DNA for early infant diagnosis using recombinase polymerase amplification. MBio 4. 2013 Apr 2; 4(2). pii: e00135–13. 18. McElgunn CJ, Pereira CR, Parham NJ, Smythe JE, Wigglesworth MJ, et al. (2014) A low complexity rapid molecular method for detection of Clostridium difficile in stool. PLoS One 9: e83808. 19. Mitarai S, Okumura M, Toyota E, Yoshiyama T, Aono A, et al. (2011) Evaluation of a simple loop-mediated isothermal amplification test kit for the diagnosis of tuberculosis. Int J Tuberc Lung Dis 15: 1211–1217. 20. Niemz A, Boyle DS (2012) Nucleic acid testing for tuberculosis at the point-ofcare in high burden countries. Expert Reviews in Microbiology. Sep; 12(7): 687– 701.
1. Niemz A, Ferguson TM, Boyle DS (2011) Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol 29: 240–250. 2. World Health Organization (2007) Early detection of HIV infection in infants and children. Guidance note on the selection of technology for the early diagnosis of HIV in infants and children. 1–11. 3. Ciaranello AL, Park JE, Ramirez-Avila L, Freedberg KA, Walensky RP, et al. (2011) Early infant HIV-1 diagnosis programs in resource-limited settings: opportunities for improved outcomes and more cost-effective interventions. BMC Med 9: 59. 4. Newell ML, Coovadia H, Cortina-Borja M, Rollins N, Gaillard P, et al. (2004) Mortality of infected and uninfected infants born to HIV-infected mothers in Africa: a pooled analysis. Lancet 364: 1236–1243. 5. Persaud D, Gay H, Ziemniak C, Chen YH, Piatak M Jr, et al. (2013) Absence of detectable HIV-1 viremia after treatment cessation in an infant. N Engl J Med 369: 1828–1835. 6. Almassian DR, Cockrell LM, Nelson WM (2013) Portable nucleic acid thermocyclers. Chem Soc Rev 42: 8769–8798. 7. Byrnes S, Fan A, Trueb J, Jareczek F, Mazzochette M, et al. (2013) A Portable, Pressure Driven, Room Temperature Nucleic Acid Extraction and Storage System for Point of Care Molecular Diagnostics. Anal Methods 5: 3177–3184. 8. Pai NP, Pai M (2012) Point-of-care diagnostics for HIV and tuberculosis: landscape, pipeline, and unmet needs. Discov Med 13: 35–45. 9. Roskos K, Hickerson AI, Lu HW, Ferguson TM, Shinde DN, et al. (2013) Simple system for isothermal DNA amplification coupled to lateral flow detection. PLoS One 8: e69355. 10. LaBarre P, Gerlach J, Wilmoth J, Beddoe A, Singleton J, et al. (2010) Noninstrumented nucleic acid amplification (NINA): instrument-free molecular malaria diagnostics for low-resource settings. Conf Proc IEEE Eng Med Biol Soc 2010: 1097–1099.
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27. Mohon AN, Elahi R, Khan WA, Haque R, Sullivan DJ, et al. (2014) A new visually improved and sensitive loop mediated isothermal amplification (LAMP) for diagnosis of symptomatic falciparum malaria. Acta Trop 134C: 52–57. 28. Tomita N, Mori Y, Kanda H, Notomi T (2008) Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3: 877–882. 29. Kersting S, Rausch V, Bier FF, von Nickisch-Rosenegk M (2014) Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis. Malar J 13: 99. 30. Clouse KA, Powell D, Washington I, Poli G, Strebel K, et al. (1989) Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone. J Immunol 142: 431–438. 31. Kim HJ, Tong Y, Tang W, Quimson L, Cope VA, et al. (2011) A rapid and simple isothermal nucleic acid amplification test for detection of herpes simplex virus types 1 and 2. J Clin Virol 50: 26–30. 32. Sheffield J, Goteti G, Wood EF (2006) Development of a 50-yr high-resolution global dataset of meteorological forcings for land surface modeling. Journal of Climate 19: 3088–3111. 33. Kalnay E, Kanamitsu M, Kistler R, Collins W, Deaven D, et al. (1996) The NCEP/NCAR 40-Year Reanalysis Project. Bulletin of the American Meteorlogical Society 437–471.
21. Ratliff AE, Duffy LB, Waites KB (2014) Comparison of the illumigene Mycoplasma DNA Amplification Assay and Culture for Detection of Mycoplasma pneumoniae. J Clin Microbiol 52: 1060–1063. 22. Surabattula R, Vejandla MP, Mallepaddi PC, Faulstich K, Polavarapu R (2013) Simple, rapid, inexpensive platform for the diagnosis of malaria by loop mediated isothermal amplification (LAMP). Exp Parasitol 134: 333–340. 23. Curtis KA, Rudolph DL, Nejad I, Singleton J, Beddoe A, et al. (2012) Isothermal amplification using a chemical heating device for point-of-care detection of HIV1. PLoS One 7: e31432. 24. Hatano B, Maki T, Obara T, Fukumoto H, Hagisawa K, et al. (2010) LAMP using a disposable pocket warmer for anthrax detection, a highly mobile and reliable method for anti-bioterrorism. Jpn J Infect Dis 63: 36–40. 25. Huang S, Do J, Mahalanabis M, Fan A, Zhao L, et al. (2013) Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis. PLoS One 8: e60059. 26. Chow WH, McCloskey C, Tong Y, Hu L, You Q, et al.(2008) Application of isothermal helicase-dependent amplification with a disposable detection device in a simple sensitive stool test for toxigenic Clostridium difficile. J Mol Diagn 10: 452–458.
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Abstract Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25–43uC, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10uC and rarely above 43uC. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31uC and 43uC. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30uC. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10uC and 30uC. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15uC, 20uC, 25uC and 30uC. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in LRS. Citation: Lillis L, Lehman D, Singhal MC, Cantera J, Singleton J, et al. (2014) Non-Instrumented Incubation of a Recombinase Polymerase Amplification Assay for the Rapid and Sensitive Detection of Proviral HIV-1 DNA. PLoS ONE 9(9): e108189. doi:10.1371/journal.pone.0108189 Editor: Cecilio Lo´pez-Galı´ndez, Centro Nacional de Microbiologı´a - Instituto de Salud Carlos III, Spain Received April 29, 2014; Accepted August 20, 2014; Published September 29, 2014 Copyright: ß 2014 Lillis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: This work was supported by the United States National Institutes of Health, National Institute of Allergy and Infectious Diseases (http://www.niaid.nih. gov/Pages/default.aspx)via award number R01AI097038. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: OP and MP are employees are of TwistDx Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors have no competing interests. * Email: [email protected]
as p24 antigen detection or reverse transcriptase activity [2]. However, the infrastructure and equipment requirements to perform PCR-based HIV diagnostics results in the need to ship specimens from rural clinics to centralized facilities for testing. In low income countries, shipping and the logistics of reporting results can delay test results for months, often contributing to loss to follow up of confirmed HIV infection [3]. The outcome for HIV infected infants who are left untreated is very poor and over 50% of HIV infected infants die within 2 years if left untreated [4]. By contrast, rapid treatment can potentially lead to a reduced viral reservoir and better outcomes, further emphasizing the need for rapid point of care diagnostics [5]. In the last decade, an expanding variety of novel NAATs have been developed to specifically meet the challenges of performing diagnostics outside of well-equipped facilities in low resource settings (LRS) [1,6–9]. A particular area of research has been the
Introduction The application of nucleic acid amplification technologies (NAAT) has had a significant and beneficial impact on the diagnosis of infectious diseases. While a wide variety of molecular based technologies have been developed to accurately detect a broad range of clinically relevant pathogens in just a few hours [1], the majority of these are designed for use in fully equipped laboratories with good infrastructure, reliable electrical supply and with highly trained staff to operate them [2–7]. Therefore, there remains a critical need for high quality diagnostics that can be performed closer to the point of care in low resource settings (LRS) to aid in the clinical diagnosis and subsequent treatment of endemic diseases such as HIV, tuberculosis and malaria. PCRbased assays are typically used for early infant diagnosis (EID) of HIV, as they are more sensitive than other existing methods such PLOS ONE | www.plosone.org
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recently a group reported the sensitive detection of Plasmodium falciparum using an RPA assay and minimal instrumentation [29]. The use of ambient temperature or simple chemical heaters for an RPA-based diagnostic has not yet been evaluated. We previously described the development of an RPA assay for rapid diagnosis of infant HIV that showed high sensitivity and specificity to the major subtypes of HIV-1 circulating globally using electrical powered, laboratory-based instrumentation [17]. In this study we investigated the potential for ambient temperatures to incubate this HIV-1 RPA assay in the absence of a reliable power source. We also analyzed meteorological data to understand the seasonal temperature fluctuations across sub Saharan Africa, the primary target market of our intended HIV diagnostic, to better understand if ambient temperatures could reliably support RPA assay use across this region. In addition, to incubate RPA assays when ambient temperatures are below 30uC, we developed a simple, low cost ($0.10 US) and reusable prototype chemical heater. Our findings in this study suggest that it is possible to develop affordable and effective incubation platforms for an RPAbased diagnostic for use in the most resource limited of settings.
development of isothermal amplification methods for nucleic acid based diagnosis. These technologies employ a uniform temperature to amplify targeted nucleic acids [1]. When compared to current PCR assays, the use of isothermal technologies reduces the need for high precision instrumentation, consistent electrical power and complex sample preparation [10,11]. Such amplification methods include loop mediated amplification (LAMP) [12], nucleic acid sequence based amplification (NASBA) [13], cross priming amplification (CPA) [14], helicase dependent amplification (HDA) [15] and recombinase polymerase amplification (RPA) [16]. In support of these amplification technologies, several electricity dependent instruments are now available to incubate the assays and detect amplification [6,18–22]. Although some of these are battery powered, they still require some degree of sustainable electrical power for recharging, and their cost (while much less than large or mid-sized real time PCR instruments) may limit their widespread adoption for use in many peripheral settings. Cheap but robust incubation systems, such as the noninstrumented incubation amplification (NINA) platform, employ chemically derived heat for assay incubation; greatly reducing costs and increasing the potential for use at the periphery [11]. These platforms have been demonstrated to effectively amplify LAMP and HDA assays [23–25]. Detection of amplification products with NINA relies upon visual inspection of the test reaction for reactivity or the use of a sealed immunochromatographic strips to capture and detect hapten labeled amplicons [26– 28]. Simple yet effective technologies like these enhance the potential to use isothermal assays and introduce high performance NAATs to the most austere environmental settings where electrical power is often sporadic or absent. RPA is an isothermal amplification method that utilizes a recombinase to facilitate the insertion of oligonucleotide primers into their complement in a double-stranded DNA molecule [16]. The use of opposing primers allows the exponential amplification of a defined region of DNA in a manner similar to PCR. Detection of HIV RPA amplicons relies on the use of an oligonucleotide probe with a specific abasic nucleotide analogue that is recognized and cleaved by an endonuclease only when the probe is bound to its complementary target sequence [16]. The amplified products can typically be detected after only 5–20 minutes, depending on amplicon size and the template copy number and also on the sensitivity of the detection method [16]. This time to test result is significantly more rapid than other isothermal assays which generally have incubation times in the range of 45–60 minutes [12–15]. RPA is also unique compared to many other isothermal amplification methods in that it has an incubation temperature range similar to ambient temperatures ranges found south of 20uN in continental Africa and has been reported to amplify DNA across a broad range of incubation temperatures ranging from 25– 43uC using the standard reaction conditions and formulation [16]. Moreover, in contrast to the other isothermal NAAT technologies, RPA does not primarily rely on physio-chemical dependent hybridization of oligonucleotides for functionality, but rather operates by an enzymatically driven primer-binding process. For this reason RPA does not require the reaction temperature to be precisely controlled during the reaction to be specific and an assay will tolerate comparatively large variance in the incubation temperature without apparent loss of overall sensitivity or specificity. These features translate to reduced power requirements and less stringent temperature control, further reducing the complexity and cost of an incubator. The use of simpler heating devices has been explored in previous studies. For example, PLOS ONE | www.plosone.org
Materials and Methods RPA amplification and Detection TwistAmp nfo RPA reactions were supplied by TwistDx Ltd., Cambridge, United Kingdom. HIV pol RPA primers and probe, described previously [17], were added to the RPA nfo assays. 10 copies of HIV DNA were added to each of 3 replicate reactions at each temperature tested. HIV proviral target DNA was from the ACH-2 cell line that contains a single full-length integrated copy of HIV-1 Bru (clade M, subtype B; GenBank accession number K02013.1) DNA per cell [30] and was prepared and quantified as previously described [17]. Negative template controls (NTC) contained nuclease free water in place of DNA. All TwistAmp reaction mixtures were prepared in a final volume of 50 mL as previously described [17]. Incubation was performed at the test temperature for either 20 or 30 minutes, with brief mixing by inverting 3 times at 5 minutes. The RPA nfo reactions were immediately placed on ice to slow the reaction and limit the effect of the slight differences in incubation times before the sequential addition of 5 mL EDTA (250 mM) to halt each reaction. Detection of hapten-labeled RPA amplicons was assessed with an immunochromatographic strip (ICS) fully enclosed in a cassette (BESt Cassette II; BioHelix Corp., Beverly, MA) [31]. Amplification products were also analyzed by gel electrophoresis.
RPA incubation To mimic various ambient temperatures, all test reaction components, RPA reagents, aluminum blocks machined to hold 0.2 mL PCR tubes, and sodium acetate trihydrate (SAT) heaters were pre-incubated for 2 hours prior to reaction set up in an environmental control chamber (Coy Laboratory Products Inc., MI, USA; or Associated Environmental Systems, Ayer, MA, USA) at temperatures between 10–44uC. Three thermocouples in each environmental chamber confirmed the temperature prior to reaction set up. Humidity in all experiments was at levels between 60–65%. The controlled incubation of RPA assays was performed using a conventional PCR thermocycler (Applied Biosystems, Foster City, CA, USA).
Exothermal heaters Sodium acetate trihydrate (SAT) (Sigma Aldrich, St Louis, MO, USA) mixtures at concentrations of 70%, 72.5% and 75% were prepared and liquefied by heating to 70uC, and aliquoted into 2
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either 15 mL screw cap tubes (Fischer Scientific, Pittsburg, PA, USA) or 22 mL lidded plastic souffle´ cups (Dart Container Corporation, MI, USA) with insulating foam (McMaster-Carr, Elmhurst, IL, USA) cut to fit each receptacle (Figure 1). A 5 mm diameter punch was made in the center of each cup lid to allow the insertion of a 0.2 mL PCR tube containing RPA reactions. Heat release via crystallization of the liquid SAT mixtures was induced by seeding with powdered SAT. Solidified SAT reactors were heated to 80uC for 20 minutes to reliquefy the SAT mixture, by either placing into a water bath or incubator heated to 80uC.
Results HIV-1 RPA assay performance at temperatures from 15uC to 44uC To determine the range of temperatures that our HIV-1 RPA assay can tolerate, we tested the ability of this specific RPA assay to amplify 10 copies of HIV DNA at a range of temperatures from 15uC to 44uC using ICS cassettes to detect RPA amplicons. Initially, we assessed this temperature range under precisely controlled conditions using a thermocycling machine. For assays incubated for 20 minutes at #30uC, performance of the HIV-1 RPA assay on 10 copies of HIV-1 proviral DNA was not optimal, with only 3 temperatures producing any positive results (1/3 at 15uC and 29uC, or 2/3 at 30uC), and all 3 replicates failing at temperatures between 20uC and 27uC (Table 1). The lower incubation temperature reduces enzyme activity leading to limited amplicon formation after 20 minutes and is not be enough to be detected by LFS; however as shown by the 1 positive sample in the reactions incubated at 15uC there is remains an occasional possibility of sufficient product being amplified for detection. When incubation times were increased to 30 minutes, performance improved at temperatures between 27uC and 30uC, with all 3 replicates being positive for each temperature, but not at temperatures of 25uC and below (data not shown). Even longer incubation times than 30 minutes, which may have resulted in positive RPA signals at low temperatures, have not been tested in this study. Incubation temperatures between 31uC to 43uC provided optimal conditions in which all 3 replicates were positive at each of the 8 temperatures tested (24/24, 100%; Table 1). When increased to 44uC, performance was impaired with only 1 of 3 reactions positive. There were no false positives at any of the temperatures tested (0/15, Table 1). Gel electrophoresis was also performed on reactions from each temperature (Data not shown). Amplification products of the expected size (,150 bp) were observed in all positive reactions heated at temperatures from 27uC–44uC. No amplification products were observed in reactions that gave negative results incubated below 27uC. This indicates that the negative results observed at lower temperatures were due to limited or no production of RPA amplicons presumably due to reduced enzyme activity and not due to competition from nonspecific product formation. We next tested our HIV RPA assay using an environmental chamber to simulate reactions incubated at ambient temperatures between 15uC and 44uC. Results were very similar to the thermocycler incubation results. Incubation for 20 minutes at temperatures #30uC were suboptimal, with only 2 of 3 reactions at 29uC, and 0 of 3 reactions positive at all other temperatures # 30uC (Table 1). When incubation times were increased to 30 minutes, 3 of 3 reactions performed at 30uC were positive, with all other results unchanged (data not shown). Incubation at ambient
Temperature measurement Ambient temperatures and thermal profiles within the SAT solutions were measured using T-type – Physitemp IT-23 thermocouples (Omega Engineering, Everett, WA, USA) and a NI 9211 data logger (National Instruments, Redmond, WA, USA). Prior to use, each thermocouple was calibrated in boiling distilled water to verify a 100uC temperature reading on the data logger. For ambient temperature assessment within the environmental chambers, one thermocouple was positioned beside the 0.2 mL reaction tube within the tube rack, and one next to the tube rack. As a proxy for reaction temperature, an additional thermocouple was inserted through a 1.27 mm diameter hole in the lid of a 0.2 mL PCR tube filled with 50 mL deionized water which represents the RPA reaction volume. When incubating RPA reactions in the SAT heaters, a thermocouple was positioned beside the 0.2 mL reaction tube (Figure 1). The temperature was recorded at a rate of 1 data point per second for the duration of each experiment. Temperature data was further analyzed and displayed using Microsoft Excel (Microsoft Corp., Redmond, WA, USA).
Air temperature data A previously published bias-corrected meteorological dataset for Africa was analyzed to determine the range of daily ambient temperatures expected [32]. The dataset is derived from meteorological analyses of the physical state of the atmosphere that are produced every three hours using a weather forecasting model. The horizontal resolution of the dataset is 161 degree latitude/longitude (approximately 1006100 km) [33]. Air temperature data are bias and elevation corrected with qualitycontrolled meteorological data from stations in Africa, and the diurnal cycle of surface air temperature is adjusted to provide the best match with meteorological station data [32]. These corrections remove a bias of 2–4uC in surface air temperature over the African continent. The dataset is available for the years 1948– 2010, but we use data only from the period 2000–2010 because the statistics were found to be insensitive to the inclusion of additional years.
Figure 1. The prototype SAT heater used to incubate RPA reactions. (A) A photograph of an activated SAT heater, the reaction tube is filled with blue dye to improve definition. (B). A cross sectional diagram depicting the SAT heater and other test components. doi:10.1371/journal.pone.0108189.g001
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0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
2/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
0/3
0/3
0/3
0/3
0/3
0/3
209
2/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
2/3
0/3
0/3
0/3
0/3
309
209
309
209 0/3
HIV
NTC
HIV
1/3
Ambient Temperature
Thermocycler
15
Temp (6C)
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
209
NTC
Table 1. A comparison of the performance of HIV RPA assays with reaction incubation in either a thermocycler or via ambient air temperature.
309
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
Amplification of HIV-1 DNA Using Ambient Temperature for Incubation
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Amplification of HIV-1 DNA Using Ambient Temperature for Incubation
and 75% SAT heaters, with and without insulation, to incubate HIV RPA assays from ambient temperatures of 10uC, 15uC, 20uC, 25uC and 30uC. Nine of 10 of the insulated reactions reached 95% of the maximum recorded incubation temperature in under 15 seconds (range 4 to 18 seconds, Table 2). The noninsulated reactions took longer to reach the maximum temperatures recorded, with a range of 5 to 85 seconds. At ambient temperatures of 15uC and above, insulated 75% SAT heaters rapidly generated sufficient heat to provide incubation for 30 minutes maintaining the 31–43uC temperature range required for the HIV-1 RPA assay (Table 2). At the lower ambient temperature of 10uC, the temperature fell to below 30uC in less than 10 minutes (Table 2). The 72.5% SAT heater was comparable above at ambient temperatures above 15uC, but again also did not maintain the minimal required temperature of 31uC after 20 minutes at ambient temperatures #15uC. Insulated SAT reactors containing 72.5% and 75% SAT were then assessed for their ability to incubate the HIV RPA assays at a range of ambient temperatures from 10uC–30uC in 5uC increments. As before, triplicate HIV-1 RPA assays were performed on 10 copies of HIV-1 DNA at each starting ambient temperature and SAT concentration. All 12 of 12 (100%) reactions at ambient temperatures between 15uC and 30uC were positive when incubated by either activated SAT mixture for 20 minutes, while no false positives were observed (Table 3). Starting at an ambient temperature of 10uC, all triplicate reactions failed when incubated with either the 72.5% or 75% SAT heater (Table 3). However, when repeated with increased incubation times of 30 minutes, to compensate for reduced enzyme activity at suboptimal temperatures, all 3 reactions starting at ambient temperature of 10uC were positive with either concentration of SAT tested (Table 3).
temperatures between 31uC and 43uC produced consistent results with all replicates positive in this temperature range (24/24, 100%). At 44uC, performance was once again suboptimal, with only 2 of 3 reactions positive (Table 1).
A non-instrumented heat source for incubation of RPA when ambient temperatures are #30uC The previous data suggests that an external heat source is necessary for the HIV-1 RPA assay used in this study when the ambient temperature is below 30uC. We investigated the potential to use a rechargeable, exothermic material commonly used in hand warmers, a mixture of sodium acetate trihydrate (SAT) and water. A variety of SAT concentrations (from 50% to 80% w/v in water) were assessed for the temperature profiles produced after their activation from an ambient temperature of 22uC (Table S1). This preliminary screen established that SAT concentrations of 70%–75% produced heat profiles that were above 31uC but did not exceed 45uC. Next, simulated assay temperature was measured for 30 minutes after activation of 8 mL volumes of 70%, 72.5% and 75% SAT solutions that had been stored for $2 hours prior to activation at ambient temperatures of 10uC, 15uC, 20uC, 25uC and 30uC (Figure 1). Each SAT solution was activated 32 times in individual tests at all of the ambient temperatures under consideration with average peak temperatures produced as shown in Figure 2. Maximal temperature was generally reached within 1 minute of the crystallization of the SAT mixture. The range of temperatures generated by each mixture did not exceed 45uC even when the heaters were conditioned to a simulated ambient temperature of 30uC prior to activation. However, when the ambient was reduced to 10uC, the 70% mixture failed to reach 30uC and was therefore not tested for incubation of RPA reactions (Figure 2). To test the ability to use SAT heaters to incubate the HIV-1 RPA reaction, we identified small plastic tubs that allow the RPA reaction mixtures to be placed close to the center of the activated SAT mixture (Figure 1). We assessed the performance of 72.5%
Analysis of daily and seasonal temperature data from Sub Saharan Africa To assess the viability of using RPA assays under ambient meteorological conditions, we analyzed an existing dataset of the
Figure 2. Median peak temperatures produced by different concentrations of SAT when held and activated under a variety of ambient temperatures. Ambient temperatures of SAT mixtures during activation: blue 10uC; green 15uC; orange 20uC; turquoise 25uC and red 30uC; SAT, Sodium acetate trihydrate. doi:10.1371/journal.pone.0108189.g002
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PLOS ONE | www.plosone.org 31 29 27 6 10 4
Maximum Temp. (uC)
Temp. (uC) at 10 minutes
Temp. (uC) at 20 minutes
Temp. (uC) at 30 minutes
Temp. loss over 30 minutes (uC)
Time (sec) to reach max. temp.
Time (sec) to 95% of max. temp.
No
8
82
22
13
17
26
35
18
123
9
28
31
34
37
Yes
15 No
11
89
18
21
22
30
39
5
94
5
32
34
36
37
Yes
20 No
15
57
16
24
27
33
40
5
123
3
37
38
39
40
Yes
25 No
5
111
10
31
34
38
41
4
14
3
38
39
40
41
Yes
30 No
85
287
6
37
40
42
43
6
80
8
25
27
31
33
Yes
10
7
52
21
13
17
25
34
No
75% SAT
12
109
7
30
32
35
37
Yes
15 No
7
53
16
18
20
26
34
5
111
5
35
37
39
40
Yes
20
6 3/3 3/3 3/3 3/3
15
20
25
30
309
3/3
3/3
3/3
3/3
3/3
209
0/1
0/1
0/1
0/1
0/1
309
0/1
0/1
0/1
0/1
0/1
209
3/3
3/3
3/3
3/3
0/3
SAT, Sodium acetate trihydrate; 209–20 minutes total incubation time; 309–30 minutes total incubation time; NTC - no template control; NTC, no template control. doi:10.1371/journal.pone.0108189.t003
209 0/3
Temperature (uC)
10
HIV
HIV
NTC
75% SAT
72.5% SAT
3/3
3/3
3/3
3/3
3/3
309
Table 3. The performance of HIV RPA assays when incubated in SAT reactors at a range of ambient temperatures from 10uC to 30uC.
SAT, Sodium acetate trihydrate. doi:10.1371/journal.pone.0108189.t002
Yes 33
Insulation
10
Ambient storage temp. (6C)
72.5% SAT
No
10
71
14
24
27
32
38
0/1
0/1
0/1
0/1
0/1
209
NTC
4
20
4
39
40
41
43
Yes
25
Table 2. Thermal profiles of the heat generated via the insulated and non-insulated SAT heaters under a range of ambient storage temperatures.
No
39
240
7
30
32
35
37
0/1
0/1
0/1
0/1
0/1
309
5
182
2
41
42
43
43
Yes
30 No
28
238
5
36
38
40
41
Amplification of HIV-1 DNA Using Ambient Temperature for Incubation
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Amplification of HIV-1 DNA Using Ambient Temperature for Incubation
surface air temperature in Africa between 2000–2010 [32]. Here we focus on temperatures recorded in the region of sub Saharan Africa (continental Africa south of 20uN) most afflicted by the HIV epidemic. Air temperature at any given site experiences significant variability on diurnal (day-night) to seasonal timescales. We distilled the data into 6 hourly intervals (0–6, 6–12, 12–18, 18– 24 hour local time) and examine statistics for each of four seasons (Dec-Feb, Mar-May, June-Aug, Sept-Nov). For each 161u location in the dataset, the season with the warmest mean temperature is termed the warm season and the season with the coldest mean temperature is termed the cold season. The RPA assay tests were shown to work well for the temperature range 30uC–43uC. Air temperatures in sub Saharan Africa were found to rarely exceed the upper limit (43uC) even during the warm season and the afternoon (12–18 hr local time; Figure 3A). For example, in the harshest desert regions north of 10uN, mean afternoon temperatures are below 43uC on over 70% of days. South of 10uN, where the HIV incidence is greatest and population density is higher, afternoon warm season air temperatures are lower than 43uC on over 95% of days. We examined the frequency at which the mean afternoon temperature exceeds 30uC during both the cold and warm seasons. During the cold season, the frequency of mean afternoon temperatures exceeding 30uC is lower than 10% in most locations. Exceptions include the desert regions north of 10uN where mean afternoon temperatures exceed 30uC on more than 50% of days during the cold season, and in the low-lying rainforest of the Congo Basin where mean afternoon temperatures exceed 30uC on 20–40% of days. During the warm season, a similar geographical pattern exists, but the frequency with which mean afternoon temperatures exceed 30uC is increased everywhere (Figure 3B). North of the equator during the warm season, mean afternoon temperatures exceed 30uC on over 50% of days. For most noncoastal locations south of 10uS, warm season mean afternoon temperatures exceed 30uC on approximately 60% of days. In the Congo Basin, warm season mean afternoon temperatures exceed 30uC on more than 40% of days. Exceptions include strongly elevated terrain (.2000 m) where mean afternoon temperatures rarely exceed 30uC. Analysis of the surface temperature data show that the mean afternoon air temperatures over most of subSaharan Africa exceed 10uC on nearly all days, even during the cold season (Figure 3C), so that a simple heating system should lead to successful RPA tests at most locations under almost all ambient environmental conditions.
Discussion RPA provides an attractive method for use in point of care diagnostics because it can be performed over a relatively broad range of incubation temperatures with no observed loss in performance when end point detection of the assay product was used. In this work we assessed the performance of a previously described HIV-1 RPA assay to reliably detect 10 copies of HIV DNA using ambient temperatures between 30uC–43uC, using the standard, commercially available formulation of RPA reagents. At ambient temperatures below 31uC, an exothermic chemical heater could be used to provide supplementary heat for optimal RPA performance to as low an ambient temperature of 10uC. These results demonstrate that, even in the absence of electricity or a battery dependent heater, RPA is significantly faster than other isothermal assays that are being developed as tools for NAAT based diagnostics in LRS. The incubation time required was only 20 minutes at the higher temperatures, with an increase to 30 minute incubations necessary at the lower temperature of 30uC to compensate for reduced enzyme kinetics at suboptimal temperatures. Longer incubation times have been shown to produce positive results at incubation times below 30uC when used with other unrelated RPA assays but longer incubation times were not tested in this study as this would lead to a less acceptable time to result. To reinforce the plausibility of using ambient temperature for RPA incubation we performed an analysis of existing meteorological data for the sub Saharan Africa region, the center of the HIV epidemic, to determine the daily ambient temperature ranges in the warmest and coldest seasons. In the warm season, the entire region has surface temperatures that often exceed 30uC after noon, providing potential to incubate RPA assays without the need for a heater during this season in many areas within this region. In addition, pooled temperature data revealed that average temperatures were rarely observed to elevate above 43uC in this region, and therefore temperatures too high for optimal RPA are not commonly encountered. However, the analysis of seasonal meteorological data highlighted that many regions in sub Saharan Africa have ambient temperatures below 30uC, especially in elevated regions which represent a significant area of this landmass. From analysis of the meteorological data we also noted that ambient temperatures of less than 10uC are not commonly observed in this region. While this work focused on sub-Saharan Africa where the HIV epidemic is greatest and so is the primary intended market for the HIV diagnostic, there are also other LRS worldwide which would
Figure 3. Maps depicting sub Saharan Africa and the frequency (fraction of days) on which mean afternoon (12–18 hr local time) surface air temperatures are below 436C during the warm season (A), exceed 306C in the warm season (B) and are above 106C in the cold season (C). doi:10.1371/journal.pone.0108189.g003
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Amplification of HIV-1 DNA Using Ambient Temperature for Incubation
greatly benefit from better access to RPA based NAATs. While the climate might differ to those found in Sub Saharan Africa, many will have day time ambient temperatures that will allow for incubation using the approaches discussed herein. To accommodate the lower temperatures observed we developed basic chemical heaters that can successfully incubate HIV-1 RPA assays even when the ambient temperature is as low as 10uC. These prototype SAT heaters were rechargeable by heating at 80uC for 20 minutes and cost less than $0.10 (USD) to make per prototype device. In areas where the temperatures fall below 10uC the SAT concentrations may be further adjusted to create a heater that will incubate the RPA reactions to within the required temperature ranges. Insulation of these heaters will be vital to minimize significant heat loss. For those with ambient temperatures above 45uC we would recommend that testing is held off until temperatures lie within in a more acceptable range. The SAT heater used in this study is only a prototype, and not the final design. It is anticipated that the final SAT heater device will be fully enclosed, with metal kinetic switch housed within the solution to initiate activation. The device can then be easily placed in a cup of boiled water to resolubilize the SAT before it is ready to be used again. Ambient temperatures and SAT heaters are not the only instrument free options available. In addition, it is interesting to note that human body temperature is within the optimal temperature range for our HIV-1 RPA assay, and suggests that manual incubation may be possible. A simple device could be created, which could be placed under the arms or held to incubate the tube to approximately 35–37uC and allow for amplification to occur. Overall, there is compelling evidence that RPA can be used for the application of high performance NAAT diagnosis of HIV-1 as well as other infectious diseases using either the ambient environmental temperature if above 30uC, or by using the most basic of heat sources for incubation when the ambient temperature is greater than 10uC. NAAT-based diagnostic tests in low resource settings face a variety of challenges, including access to adequate electrical power to run equipment or to recharge batteries, as many of these NAATs require prolonged incubation at elevated
temperatures (.55uC) within a narrow range for optimum amplification. Typically this means that powered instrumentation is necessary to address precise incubation temperatures. However RPA does not have such stringent requirements. A previously described RPA assay to detect methicillin resistant Staphylococcus aureus was similarly noted to detect target DNA in a temperature range spanning from 25uC to 44uC, [16], therefore it is highly probable that this temperature range could be applied to any RPA based target. This relatively broad range of incubation temperatures suggests that RPA can be effectively performed using either ambient temperatures without a heat source or by using a simple electricity-free heat source supplied via an exothermic chemical reaction [29]. Here we demonstrate the potential benefit of using RPA in point-of-care diagnostics in settings where access to reliable electrical power is limited. As this technology does not require a precise temperature for incubation, ambient temperatures or a simple heating device can be used to achieve accurate and reproducible results, making it a powerful diagnostic tool.
Supporting Information Table S1 Thermal profiles from preliminary screening of SAT concentrations of 50%–80% (w/v) after activation from an ambient temperature of 226C. Reactions were performed in insulated 50 mL tubes. (DOCX)
Acknowledgments We thank Dr Angelika Niemz for critical edits and comments during the drafting of this manuscript.
Author Contributions Conceived and designed the experiments: DB DL OP MP LL MS JC JS RW AT. Performed the experiments: LL MS JC JS RW AT. Analyzed the data: DB DL OP MP LL MS JC PL JS RW AT JO. Contributed reagents/ materials/analysis tools: OP MP DL JO RW. Wrote the paper: LL DB DL JC MC RW JO PL JS.
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21. Ratliff AE, Duffy LB, Waites KB (2014) Comparison of the illumigene Mycoplasma DNA Amplification Assay and Culture for Detection of Mycoplasma pneumoniae. J Clin Microbiol 52: 1060–1063. 22. Surabattula R, Vejandla MP, Mallepaddi PC, Faulstich K, Polavarapu R (2013) Simple, rapid, inexpensive platform for the diagnosis of malaria by loop mediated isothermal amplification (LAMP). Exp Parasitol 134: 333–340. 23. Curtis KA, Rudolph DL, Nejad I, Singleton J, Beddoe A, et al. (2012) Isothermal amplification using a chemical heating device for point-of-care detection of HIV1. PLoS One 7: e31432. 24. Hatano B, Maki T, Obara T, Fukumoto H, Hagisawa K, et al. (2010) LAMP using a disposable pocket warmer for anthrax detection, a highly mobile and reliable method for anti-bioterrorism. Jpn J Infect Dis 63: 36–40. 25. Huang S, Do J, Mahalanabis M, Fan A, Zhao L, et al. (2013) Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis. PLoS One 8: e60059. 26. Chow WH, McCloskey C, Tong Y, Hu L, You Q, et al.(2008) Application of isothermal helicase-dependent amplification with a disposable detection device in a simple sensitive stool test for toxigenic Clostridium difficile. J Mol Diagn 10: 452–458.
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